The skin is a non-reproductive, oestrogen-responsive tissue via specific receptors; however their mechanisms of action involving through these nuclear subtype receptors in the genomic and non-genomic pathway remain unclear. Moreover new advances in the development of selective ligands for ERÎ± and ERÎ² and other transcriptional activators of oestrogen signaling may provide more insight understanding of the processes involved in impaired oestrogen action on human skin. This may include the role of cell migration in a variety of biological processes; such as immune responses, embryonic development, cancer metastasis and homeostasis & wound healing, (Lauffenburger and Ridley.; et al. 1996, 2003). This could have beneficial implications in many skin therapies. Cytoskeleton remodeling and disassembly of focal adhesion sites facilitates cell migrations, as a result these lead to formation of membrane protrusions i.e. lamellipodia, filopodia and tractions forces that permit cell movement (Mitra et al., 2005). Cell migration is fundamental to wound healing and the scratch wound assay is inexpensive and a commonly used method to study cell migration in vitro (Lampugnani, M.G, 1999).
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Human keratinocytes are the primary cells and is a major component of the epidermis, comprising 95% of the cells (McGrath, J.A., et al.), they are specialised cells that synthesise keratin. Keratinocytes play an important role in wound healing and these results indicate oestrogen has effect on proliferation and migration of human keratinocytes. Human keratinocytes can express both ERÎ± and ERÎ² (Verdier S. et al.; 2004) and these effects may appear independently. The expressions of both these receptors have been previously demonstrated. In vivo studies indicate that administration of ERT can have beneficial effects on aging-related skin deterioration including cutaneous wound healing in post menopausal women (Ashcroft. et al., 2002). Therefore the aim of this project was to determine the effects of 17Î²-E2, selective ERÎ± and ERÎ² agonists on cell migration in cutaneous wound healing under varying dose response and time course.
5.1. The effect of oestrogen, PPT and DPN on keratinocyte cell migration status.
My findings strengthen the concept of the non-genomic signaling of oestrogen receptors in the determination of cell migration. This in vitro study of a precursor steroid hormone; oestrogen demonstrated that oestrogen can directly effect cell migration of human keratinocyte cells in an in vitro wound healing assay. This suggests that oestrogen maybe able to stimulate migration via ERÎ±, following conversion from DHEA. Additionally the effect of oestrogen on proliferation was also observed. This would result in over confluence and clumping of cells which would suggest the cells are dividing (figure 3b). Increased migration was observed in response to 17Î²-E2 and the selective ERÎ± and ERÎ² agonist, implying that stimulation may occur via ERÎ± and ERÎ² depending on the length of exposure and dose (figure 4). Cell migration to heal wound were significantly increased in vitro (figure 4), at specific doses. PPT increased cellular migration response towards wound closure greater in comparison to DPN following administration of 1nM PPT (figure 4F). In contrast, at 48hours wound closure were increased by DPN at 100nM (figure 4d). Following post-treatment with 10nM and 1000nM had no apparent impact of agonist activity on keratinocyte migration in vitro. This data can now confirm that both selective agonists ERÎ± (PPT) and ERÎ² (DPN) are expressed in human keratinocytes in vitro and that both contribute independently to stimulate the action of keratinocyte proliferation and migration following long term exposure to 17Î²-E2, though their actions may be dependent to dose and time. This data follows coherently with the findings in the recent report on the involvement of both these receptors in the positive effects of E2 in keratinocyte following trauma-hemorrhage (Moeinpour et al., 2008).
The study demonstrated that wound healing was observable post-treatment between 24-48 hours following wounding. It was more informative to observe a section of the wound at high magnification (X10) rather than entirety of the well at (X4) as at X10 cell morphology, migration and orientation was observable. Significant cell migration could be seen at early time point, 6 hours with lamella and protrusions at the wound margin, alternatively at mid-point, 24hrs cell migration could be observed easily at low magnification (X4) and after late-time point's, 30-48hrs the wounds were completely healed (figure 3). A control well supplemented with 0.0001% ethanol showed cell polarisation at early-point's 3-6hrs of the non-migrating cells. However migration of the cell sheet with neighbouring cells was concerted following 24-48hrs as they moved together in the wound (figure 3a).
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The process of wound healing begins as the cells polarise towards the wound, initiate protrusion, migrate and finally close the wound. These data implicate that long-term administration of oestrogen increases the extent of wound healing by reducing the wound size in human skin. The major limitation for scratch wound assay is the lack of wound surface space or gap between cells, that wound vary in sizes, which prevents consistent results and constructs variation from well to well. Furthermore the scratching often causes damage to the cells at the edge of the wound; this usually prevents cell migration in to the wound site and stops healing.
Furthermore a deep scratch may formulate a groove as seen in figure 4. This showed downward cell migration rather than across the wound bed. The actual movement is usually very slow. The surface grooves usually align the cells and steer the cells to migrate across. This surface topography has a considerable effect on migrate rate and is critically influenced by cell shape morphology, direction of migration and cells orientations. Cells clustering observed in the wells would suggest they are dividing and decreased migration or aberrant morphology was showed to increase in the number of mitotic cells, which manifest as bright spheres (figure 3b). These dividing cells would have moved into the wound surface and appear as rounded cells. These dividing cells could be further shown by starving with proliferation markers. Furthermore the change in cellular shape morphology as seen in figure 4, suggests that these dendritic-like cells behind the wound will migrate from the back of the migrating cells. This shows that the shape of a moving cell may vary noticeably, but its leading front has a characteristic behavior that generally pulls the cell forward (figure 4). These cells would have migrated across the surface of the wound by extending filopodia from the leading front of the wound edge. Furthermore the addition of growth factors can be used to stimulate the formation of filopodia which will direct keratinocyte division and wound closure (Bentley and Toroian-Raymond., 1986).
Finally to sum up, overall these results would suggest that oestrogens could maintain and/or increase keratinocyte responsiveness to ERT via modulating the level of its receptor and that keratinocyte proliferation and migration may be adversely affected by oestrogen deprivation as in post-menopausal. Therefore this statement provides a clear indication of the role of oestrogen importance in cutaneous wound healing and that in its absence significant delay in healing may be as a result (Jorgensen and Schmidt., 1962). It has been proven that HRT/ERT improve wound healing and age-related skin deterioration, however the effect and efficacy of oestrogen and agonists may be varied within other anatomical sites. These may be expressed differentially within specific anatomical sites, an example of over-expression of ERÎ± action is satisfied in the implication of breast cancer in human breast skin. 76% of breast cancers are registered as ER+, therefore an important diagnostic tool could include the assessment of oestrogen receptor status within human breast skin (Crowe et al., 1991). Furthermore Verdier-Sevrain et al, found that keratinocytes derived from normal foreskin can express both ERÎ± and ERÎ² in the presence of phenol red culture (mimics oestrogen activity), however studies by Kanda and Watanabe observed the findings of ERÎ² and not ERÎ± (Kanda and Watanabe, 2003) in the phenol red-free medium. Indeed my experiment showed oestrogen to directly stimulate migration via ERÎ±, in keratinocytes. These findings are consistent and a possible explanation for this distinct finding may be due to different cultural conditions and cells used from different anatomical sites.
In conclusion I have fulfilled the objective of the study; the present data indicate function for ERÎ± and ERÎ² mediating cellular proliferation and migration in vitro keratinocytes. This study has shown that keratinocyte are oestrogen responsive cells that display high oestradoil binding affinity and can express both known receptors in cutaneous wound healing. In addition oestrogen can normally modulate epidermal function in premenopausal women and that oestrogen supplementation may improve skin quality and function in post-menopausal women following keratinocyte proliferation and migration (Verdier S. et al.; 2004). Although several in vivo and in vitro studies have shown the positive effect of oestrogen on skin wound healing, the mechanism underlying the relative actions of ERÎ± and ERÎ² still remain unclear. Therefore further understandings of oestrogen and involvement of its receptors may have increasing interest and alternative approaches in skin wound healing.
5.2. Future work.
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Unfortunately this study was limited to a specific cell line and the dose response and time course was at maximum of 2 days. Furthermore the observation period was long and limited to six different time points and four varying doses. Although this may have been sufficient considering the number of tests required, it could not be considered an excessive length of time. It can therefore be assumed that these results may be applicable only to in vitro cell line. On the other hand my study was performed under phenol-red free cultural condition; another test to support the effect of oestrogen receptors on keratinocyte migration in the presence of phenol red could be beneficial. Although the objective of the study was to establish cell migration, additionally cell proliferation was also present. Before it can be definitively noted that oestrogen and selective agonists have an impact upon cell migration, the proliferative and migratory effects would need to be investigated independently. To determine whether migration or proliferation was induced; the migration assay could have been repeated with a proliferation assay with the addition of a proliferation marker and mitomycin C (MMC). This causes alteration in cells morphology and function (block genomic pathways) so the proliferative and migratory effects of oestrogen on keratinocytes can be separated. Furthermore as mentioned, the limitation of this study to a specific epithelial cell line keratinocyte, NCTC. I would recommend that the oestrogen and agonists are to be exposed to different anatomical sites as it is not uncommon for these to have other effects within these sites.